Mammalian phosphatidylinositol-specific phospholipases C (PI-PLC) are key effectors of the action of growth factors, neurotransmitters, and hormones. These enzymes respond to stimulation of extracellular receptors by increasing hydrolysis of phosphatidylinositols and generation of intracellular second messengers, diacylglycerol and inositol phosphates. On the other hand, the bacterial PI-PLCs have no confirmed physiological function, but due to their small size, good stability and analogous chemical mechanism to mammalian enzymes, they can serve as convenient models of more complex mammalian enzymes. The available results indicate that modulation of PI-PLC activity occurs at two levels, by interaction with the lipid-water interface and with the hydrophobic monomolecular substrate, and that conformational changes of enzyme and substrate are involved. The goal of the proposal is to understand the nature of those changes and their effect on the catalytic mechanism. This goal will be achieved by studying conformational changes of the enzyme and hydrophobic substrate analogs upon progression from the ground state complex to the complex of enzyme with the transition state. The enzyme from Bacillus thuringiensis, expressed in E. coli, will be the focus of this project. The following specific aims will be completed: (1) Several types of conformationally flexible, cleavage-resistant analogs of phosphatidylinositol will be synthesized. The phosphorothioate analogs, which feature the smallest modification of the parent substrate structure, will be used as reference ligands. (2) Conformationally constrained substrate and transition state analogs will be also synthesized. These analogs will mimic the reactive substrate conformation, and the structure of the putative transition state. (3) Structures of complexes of the enzyme with both groups of analogs will be investigated by means of multidimensional NMR, and x-ray crystallography. The latter studies will be performed by Dr. Heinz at the University of Freiburg and Dr. Williams at MRC. (4) Whether the mechanism of phospholipase C involves two-step or single-step mechanism will be studied using linear free energy relationships to assess the degree of phosphorus-oxygen bond breaking in the transition state. (5) The nature of hydrophobic and interfacial activation of PI-PLC will be investigated using mutants at positions exposed to interactions with fatty acid chains and lipid-water interface. The magnitude of hydrophobic and interfacial activation of these mutants will be determined using monomeric and micellar substrates.